J. Med. Microbiol. -Vol. 37 (1992), 310-314 0 1992 The Pathological Society of Great Britain and Ireland Sharing of virulence-associated properties at the phenotypic and genetic levels between enteropathogenic Escherichia coli and Hafnia alvei M. J.ALBERT, S. M. FARUQUE, M.ANSARUZZAMAN, M. M. ISLAM, K. HAIDER, K.ALAM, I. KABIR and R. ROBINS-BROWNE" International Centre for Diarrhoea1 Disease Research, Bangladesh, GPO Box 128, Dhaka 7000, Bangladesh and *Royal Children's Hospital, Parkville, Victoria 3052, Australia Summary. Seven strains of Hafnia alvei isolated from diarrhoea1 stools of children resembled enteropathogenic Escherichia coli (EPEC) in that they produced attaching-effacing (AE) lesions in rabbit ileal loops and fluorescent actin staining in infected HEp-2 cells. In addition, a DNA probe from a chromosomal gene required by EPEC to produce AE lesions, hybridised to chromosomal DNA from all seven H. alvei strains. These findings indicate that there is a sharing of virulence-associated properties at the phenotypic and genetic levels by H. alvei and EPEC. H . aZvei strains with these properties should be considered diarrhoeagenic. Introduction Enteropathogenic Escherichia coli (EPEC) strains are an important cause of childhood diarrhoea and characteristically are unable to produce heat-labile enterotoxin (LT) or heat-stable enterotoxin (ST), or express Shigella-like invasiveness.' However, some strains produce a shiga-like cytotoxin.2The majority of EPEC possess a plasmid of c. 60 MDa associated with localised adherence (LA) to cultured HeLa or HEp-2 cells.3 An EPEC adherence factor (EAF) probe constructed from this plasmid shows good correlation with LA in tissue-culture assay^.^ Furthermore, EPEC induce attaching-effacing(AE) lesions in the intestinal mucosa of affected animal^.^ These lesions are charac- terised by intimate attachment of bacteria to the enterocyte with the resultant destruction of microvilli, cupping and pedestal formation of plasma mem- branes, and accumulation of electron-dense fibrillar material in the terminal web adjacent to attached bacteria. The fibrillar modification probably involves polymerisation of actin filaments, which can be detected in cultured cells by a fluorescent actin staining (F AS) assay. The genes required to produce the AE lesion are located on the bacterial chromosome.' Although both LA (plasmid-borne) and AE (chromosomal) proper- ties are required by EPEC for full expression of enteropathogenicity, EPEC unable to cause LA retain the ability to cause diarrhoea, albeit less effectively than strains which possess the LA phenotype? It appears that the plasmid-associated LA factor may facilitate initial contact of the bacteria with the mucosa, after which the chromosomally encoded factor(s) produce the AE lesions.' Recently, Jerse et al. lo used transposon mutagenesis to identify a chromosomal gene that is required by EPEC strain JPNl5 (a plasmid-free derivative of EPEC strain E2348/69, serotype 0127:H6) to pro- duce AE lesions. This gene, termed eae, comprises a 28 17-bp open-reading frame capable of encoding a 102-kDa protein. A 1-kb probe derived from this region hybridised to all E. coli strains of EPEC serogroups which demonstrate AE lesions, as well as to other pathogenic E. coZi that produce these lesions, such as enterohaemorrhagic E. coli (EHEC) and E. coli RDEC-1 (an EPEC of weanling rabbits). Recently, we described a strain of Hafnia alvei (referred to as 19982 in this report), isolated from the stool of a child with diarrhoea, that produced di- arrhoea in RITARD rabbits and whole intact rabbits.'l This strain did not have any recognised conventional virulence-associated properties, such as LT, ST, shiga-like toxins (SLTs) or shigella-like invasiveness of, or adherence to, cultured HeLa cells. However, histological and electronmicroscopic exam- inations of intestinal tissues from diarrhoeic rabbits and from inoculated small intestinal loops of adult rabbits showed typical AE lesions characteristic of EPECY We have since isolated six more strains of H. alvei from stools of children with diarrhoea. We characterised these strains and investigated the sharing of virulence-associated properties with EPEC at the phenotypic and genetic levels. Received 1 Jan. 1992; accepted 27 Jan. 1992. 3 10